Assays that measure genome-wide transcript abundance or the epigenetic state of cells can reveal fundamental insights Into the regulation of the cell state, but require large numbers of cells as input. This limits their use In human immunology and In defining the heterogeneity of cell populations. The goal of our studies is to optimize and Integrate gene expression profiling and analysis of epigenetic state in rare cell populations and single cells, using the Immune response to Dengue virus and yellow fever virus (YFV) vaccination as an experimental paradigm. AIM 1: Optimize and Implement an Integrative single-cell expression profiling platform. We will optimize the Fluidigm Biomark assay to profile gene expression in single cells, and integrate single-cell expression profiling of antigen-specific B cells and T cells with single-cell analysis of VH and TCRB locus sequence. We will test whether differences in antigen-receptor sequence correlate with the differentiation state of individual cells. AIM 2: Optimize and implement epigenetic analysis of rare populations of cells We have developed a novel assay - ATAC-seq - that, for the first time, enables genome-wide profiling of chromatin accessibility In as few as 50,000 cells. We will optimize this assay and use It to survey the epigenetic landscape of rare, tetramer-sorted populations of YFV-specific CDS T cells, as well as CD4, B cell and monocyte populations. These studies will define the regulatory landscape of differentiation In each lineage. AIM 3: Integrate single-cell analysis of chromatin accessibility and gene expression. Little is known of the cell-to-cell differences In chromatin accessibility that may drive the functional and phenotypic heterogeneity characteristic of Immune cell populations. We will optimize ATAC-seq for use in single cells and combine single-cell gene expression profiling with ATAC-seq to test whether the chromatin state of single cells drives the heterogeneity In transcriptional profile across human immune cells. By extending these assays to such small cell numbers, we anticipate that this project will redefine the scope of mechanistic Information that can be obtained from the analysis of human samples.